Characterization of the Inhibitor of KappaB Kinase (IKK) Complex in Granulosa Cell Tumors of the Ovary and Granulosa Cell Tumor-Derived Cell Lines

Abstract

Granulosa cell tumors of the ovary (GCT) are a distinct, hormonally active subset of ovarian cancers. Although it has recently been shown that ∼97 % of all adult GCT harbor a novel somatic missense mutation in the FOXL2 gene, given its almost universal presence, it does not explain differences in tumor stage and/or recurrence. The nuclear factor kappaB (NFκB) transcription factor is constitutively active in two human GCT-derived cell lines, COV434 and KGN, which are useful in vitro models to investigate juvenile and adult GCT, respectively. This study aimed to determine the molecular basis and pathogenetic significance of this aberrant NFκB activity. Selective chemical inhibitors were used to target candidate components of the pathway. The constitutive activity was blocked by two independent inhibitors of IκBα phosphorylation, suggesting that aberrant activation occurs upstream of this point. NFκB inhibition resulted in a dose-dependent decrease in cell proliferation and viability and a dose-dependent increase in apoptosis. Inhibitors of earlier components of the pathway were without effect. Two independent inhibitors of inhibitor of kappaB kinase (IKK)β, a catalytic subunit of the NFκB activation complex, were unable to inhibit the constitutive activity, but surprisingly also ligand-induced activity. These findings suggest a central role for IKKβ; however, no mutations or altered expression of the IKKβ, IKKα, or IKKγ genes was observed in the cell lines or in a panel of human GCT samples. This study highlights unresolved issues in understanding the pathogenesis of GCT and in the use of the COV434 and KGN cells lines as model systems.

Tyrosine Kinase Inhibitors as Potential Therapeutic Agents in the Treatment of Granulosa Cell Tumors of the Ovary

“…14 Given the limitation of a ''squelching'' effect observed upon cotransfection with a constitutively expressing Renilla luciferase construct in the GCTderived cell lines, 14 experiments were carried out in the absence of a transfection efficiency control at least thrice with sextuplicate wells per point. All values represent the mean T SEM.…”

“…Given that abundant RAF1 mRNA expression levels have been observed in both COV434 and KGN cell lines, 14 cells were incubated with increasing concentrations of the RAF1-specific inhibitor (1Y1000 nM [data not shown]; and 1Y1000 KM) or vehicle (DMSO) alone for 24 hours, before cell proliferation and cell viability were assessed (Fig. 4).…”

“…However, current chemotherapy is based on protocols derived largely from experience with ovarian epithelial tumors, and the efficacy of these interventions remains limited. 14 As a result, approximately 80% of patients with advanced stage or recurrent tumors succumb to their disease. 13 Two human GCT-derived cell lines, COV434 and KGN, have been used as in vitro models to investigate the molecular pathogenesis of GCT.…”

“…16 Inhibition of NFJB activity using 2 independent inhibitors of IJB> phosphorylation results in a dose-dependent decrease in cell proliferation and cell viability, and a dose-dependent increase in apoptosis. 14 Two case reports of extensively pretreated, recurrent GCT responding to the tyrosine kinase inhibitor (TKI), imatinib, 17,18 suggests a potential role for these therapies in the management of GCT. On this basis, Chu et al 19 characterized the expression of the imatinib-sensitive TK genes [Mast/stem cell growth factor receptor (SCFR)/KIT, Abelson murine leukemia viral oncogene homolog (ABL), platelet-derived growth factor receptor (PDGFR)-> and -A] in human GCT samples, and examined the effect of imatinib, and subsequently nilotinib, a more potent second-generation TKI with a profile similar to that of imatinib, on COV434 and KGN cell lines.…”

Genetics and genomics of ovarian sex cord‐stromal tumors

“…Both lines are notable for constitutive activation of the NFkB and Braf/ERK signaling pathways (52)(53)(54)(55). Rosario et al (56) used a transcriptomic approach with these cell lines to identify potential targets of FOXL2.…”